While substantial progress has been made in identifying the receptors and downstream signaling mechanisms involved in taste transduction, much less is known about how taste cells transmit this information to the nervous system. Recently we identified ATP as a key transmitter linking taste cells to activation of gustatory afferent fibers. Using mice that lack the purinergic receptors P2X2 and P2X3, we showed that these mice lack gustatory responses to all taste stimuli. Further experiments by our labs and others have shown that the taste cells that express bitter, sweet, and umami taste receptors release ATP via non-vesicular mechanisms, likely hemichannels composed of Pannexin1. Further, the ATP that is released is broken down to ADP by the ecto- nucleotidase NTPDase2, expressed on the membranes of the glial-like support cells in the taste bud. However, several questions remain about the role of ATP as a transmitter in taste buds. First, is Pannexin1 required for ATP release and full activation of gustatory afferents? This has not been tested in vivo. Further, why are sour and salty responses absent in the P2X2/3 double knockout mice when the only taste cells known to release ATP are the cells that respond to bitter, sweet, and umami stimuli? Finally, what is the role of the ecto-ATPase in the perdurance of the ATP released from taste buds and how does this impact gustatory function? In this new grant proposal we will use existing knockout mice to genetically eliminate key elements of purinergic signaling in taste buds. We will use an integrated systems neuroscience approach-- from cellular assays of ATP release to gustatory nerve recording and behavior-- to answer the following questions: (1) Is Pannexin1 required for ATP release and activation of gustatory nerve fibers? We will use both a global and a conditional knockout of Pannexin1 to address whether this channel mediates ATP release in taste cells, and whether it is necessary and sufficient for full activation of gustatory afferent fibers and taste guided behavior. (2) What is the role of NTPDase2 in the perdurance of ATP release? We will utilize NTPDase2 knockout mice to determine how NTPDase2 affects the magnitude and perdurance of the ATP that is released and what role this plays in the sensitivity of the afferent nerve fibers to taste stimuli. Results from these experiments will provide important new data about the role of ATP in gustatory function. By understanding how taste cells communicate with afferent nerve fibers, it may be possible to develop pharmaceutical agents that will interfere with this process and modulate taste function. This could have an important impact on health problems that result from disorders of food intake. PUBLIC HEALTH RELEVANCE: The sense of taste evolved to allow discrimination of nutritionally important compounds from toxic substances. However, overconsumption of palatable foods can lead to obesity and Type II diabetes, now a major world heath problem. Conversely, the off-taste of a medicine can be a limiting factor in patient compliance. A better understanding of the mechanisms used by taste cells to detect chemicals and transmit taste information to the nervous system may lead to the development of pharmaceuticals that can control overconsumption and limit the bitter tastes of many drugs.